Development of a multi‐recombinase polymerase amplification assay for rapid identification of COVID‐19, influenza A and B

Abstract The coronavirus disease 2019 (COVID‐19) pandemic caused extensive loss of life worldwide. Further, the COVID‐19 and influenza mix‐infection had caused great distress to the diagnosis of the disease. To control illness progression and limit viral spread within the population, a real‐time reverse‐transcription PCR (RT‐PCR) assay for early diagnosis of COVID‐19 was developed, but detection was time‐consuming (4–6 h). To improve the diagnosis of COVID‐19 and influenza, we herein developed a recombinase polymerase amplification (RPA) method for simple and rapid amplification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2), the causative agent of COVID‐19 and Influenza A (H1N1, H3N2) and B (influenza B). Genes encoding the matrix protein (M) for H1N1, and the hemagglutinin (HA) for H3N2, and the polymerase A (PA) for Influenza B, and the nucleocapsid protein (N), the RNA‐dependent‐RNA polymerase (RdRP) in the open reading frame 1ab (ORF1ab) region, and the envelope protein (E) for SARS‐CoV‐2 were selected, and specific primers were designed. We validated our method using SARS‐CoV‐2, H1N1, H3N2 and influenza B plasmid standards and RNA samples extracted from COVID‐19 and Influenza A/B (RT‐PCR‐verified) positive patients. The method could detect SARS‐CoV‐2 plasmid standard DNA quantitatively between 102 and 105 copies/ml with a log linearity of 0.99 in 22 min. And this method also be very effective in simultaneous detection of H1N1, H3N2 and influenza B. Clinical validation of 100 cases revealed a sensitivity of 100% for differentiating COVID‐19 patients from healthy controls when the specificity was set at 90%. These results demonstrate that this nucleic acid testing method is advantageous compared with traditional PCR and other isothermal nucleic acid amplification methods in terms of time and portability. This method could potentially be used for detection of SARS‐CoV‐2, H1N1, H3N2 and influenza B, and adapted for point‐of‐care (POC) detection of a broad range of infectious pathogens in resource‐limited settings.


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and digestive tract symptoms in some patients. [2][3][4] These symptoms resemble those of patients with influenza or the common cold, resulting in misdiagnosis initially. COVID-19 rapidly spread worldwide, with 602 136492 confirmed cases reported by August 22, 2022. Since the end of 2019, COVID-19 has caused extensive loss of life and severe economic losses worldwide, 5,6 killing more than 6 455 497 people by August 22, 2022, with a mortality rate of 2%. 7 In the past two years, the mutation of the virus has accelerated its global epidemic, especially the Delta and Omicron variants, and the number of infections around the world is still increasing dramatically.
Although numerous gene amplification assays have been developed for virus detection, they are time-consuming and often suffer from poor sensitivity. [8][9][10][11] It is therefore urgent to develop an accurate, rapid point-of-care (POC) diagnosis method that can effectively identify infections and carriers to prevent the virus spreading.
To control illness progression and limit viral spread within the population, various methods (computed tomography [CT] scan, syndromic testing, nucleic acid testing and antibody testing) have been developed for early detection. 9,[11][12][13][14] Real-time reversetranscription fluorescent polymerase chain reaction (RT-PCR) of viral RNA from upper respiratory tract samples (i.e., nasopharyngeal swabs, nasal aspirates, and nasopharyngeal washes) is considered the gold-standard method for clinical diagnosis of COVID-19. 8,10,12,15 Genes encoding the nucleocapsid protein (N), the RNA-dependent-RNA polymerase (RdRP) in the open reading frame 1ab (ORF1ab) region, and the envelope protein (E) have been used to design primers and probes to detect SARS-CoV-2. For example, Corman and colleagues 15 aligned and analyzed several SARS-related viral genome sequences to design a set of primers and probes, and developed an RT-PCR method to detect SARS-CoV-2. In their study, both the E and RdRP genes achieved high sensitivity (limit of detection of 3.9 copies and 3.6 copies per reaction, respectively) for detection, whereas the N gene yielded poorer sensitivity (8.3 copies per reaction). 7 However, the RT-PCR method is time-consuming (4-6 h), labor-intensive, and instrument-dependent. Furthermore, a major challenge for RT-PCR is the difficulty in optimizing the amplification and reverse transcription steps because they occur simultaneously, which leads to lower target amplicon generation.
Loop-mediated isothermal amplification (LAMP) is a method that can amplify nucleic acids with high specificity, sensitivity, and rapidity at 60°C-65°C, that does not require expensive reagents or special instruments such as a thermal cycler. In 2003, this method was used for SARS coronavirus detection. 16 Several academic laboratories have developed and clinically tested RT-LAMP tests for SARS-CoV-2 detection. Yu and colleagues developed an RT-LAMP diagnostic platform for COVID-19, 13 but the sensitivity was low (limit of detection of 60 copies/μL), and the target region was only a fragment of ORF1ab, which may lead to failed diagnosis. The same group also reported a rapid and visual detection method for SARS-CoV-2 based on RT-LAMP 14 using primers specific for the spike protein (S) and ORF1ab genes of SARS-CoV-2, and detection could be achieved within approximately 30 min. However, this assay also suffered from low sensitivity for the S gene (2 × 10 2 copies per reaction). In addition to isothermal amplification, there are other nucleic acid tests that can be used for SARS-CoV-2 detection. For example, SHERLOCK technology is a detection strategy that uses Cas13a for RNA sensing. [17][18][19] Zhang and colleagues reported a SHERLOCK protocol for detecting SARS-CoV-2 20 involving three steps that can be completed in 1 h, starting from nucleic acid extraction, as used for RT-PCR tests for S and ORF1ab genes. However, the limit of detection ranged between 10 and 100 copies/μl of SARS-CoV-2 RNA sequence.
Clearly, more accurate, and rapid diagnostic methods are needed for diagnosis of COVID-19 during the early stages of screening.
RPA is an isothermal amplification technique for the specific, rapid, and cost-effective detection of pathogens. Due to its low operation temperature (37°C-42°C) and commercial availability of freeze-dried reagents, it has been applied outside laboratory settings in remote areas. [21][22][23][24] According to previous study, 25 two recombinase-based isothermal techniques, reverse transcription recombinase polymerase amplification (RT-RPA) and reverse transcription recombinase-aided amplification (RT-RAA), were evaluated for the detection of SARS-CoV-2 in clinical samples (e.g., 176 cases). The results showed that sensitivity of RT-RPA and RT-RAA was only 85.53% and 76.32%, respectively. In another study, one-tube SARS-CoV-2 detection platform based on RT-RPA and CRISPR/Cas12a was reported. 26 This method has high sensitivity (a low detection limit of 2.5 copies/µl input (RNA standard) and 1 copy/µl input (pseudovirus)), but the detection time is more than 50 min. An RPA and AuNP-based colorimetric assay were also be used for SARS-CoV-2 detection. 27 In their study, they can specifically target an ORF1ab and N regions of the SARS-CoV-2 genome, and bring the sensitivity of the method to one copy of viral genome sequence per test, however, there was no actual test validation with clinical samples.
What's more serious is that the mixed infection of SARS-CoV-2 and influenza A or B has caused great trouble to clinical diagnosis and treatment. The susceptibility of COVID-19 in influenza-infected people is enhanced, and the condition of mixed infection of COVID-19 is aggravated, which is easy to develop into severe pneumonia in the present work. 28,29 We developed a more convenient and faster method for detecting SARS-CoV-2, H1N1, H3N2, and influenza B based on recombinase polymerase amplification (RPA) technology. In our study, N,     Table 1. All primers were synthesized commercially (Sangon Biotech Co., Ltd.).

| Preparation of SARS-CoV-2, H1N1, H3N2, and influenza B standards
The concentration of plasmid DNA containing partial fragments of the ORF1ab, N and E genes of SARS-CoV-2 and the M, HA, and PA genes of H1N1, H3N2 and influenza B was quantified, and samples were serially 10-fold diluted from 1 × 10 6 to 1 × 10 0 copies/ml as standard templates.    Figure 1C) and last the amplification data were sent to a laptop data analysis ( Figure 1D). 3.2 | Sensitivity of the RPA assay

| Specificity of the RPA assay
To validate the specificity of the RPA approach, PBS, RNA from Influenza and B, and five COVID-19 negative samples were subjected to RPA analysis as described above. As shown in Table 2 (Table 3) and

| Strategies for SARS-CoV-2 and influenza detection by RPA
the three genes (M, HA, and PA) were used to detect the H1N1, H3N2 and influenza B, respectively. As shown in Table 3, results of RPA assay were 100% coincidence with qPCR indicated that the established RPA assay can play an important role in the detection of COVID-19 and influenza A or B.

| Validation of clinical samples
As shown in Figure 3, all 70 COVID-19 confirmed samples were identified as positive by the RPA assay.  that the detection rate of the screening method established in this study was 100% (Table 2).
These results indicate that the SARS-CoV-2, H1N1, H3N2, and influenza B RNA samples were successfully detected by the developed RPA assay. More importantly, it was found that the Cq value of RNA samples from COVID-19 patients was significantly lower than that from healthy controls ( Figure 4A,C,E). In addition, a which is also in line with China's dual target or triple target detection guidelines.
As shown in Figure 1, each experiment requires 60 μl of RNA  25 min at 39°C (Figure 1). According to the standard curves ( Figure 2), our RPA assay improved the detection limit down to 100 copies/ml for M, HA, and PA genes detection ( The results indicated that our method can be used for virus nucleic acid detection of coinfected samples.
As shown in Figure 3 and Table 2, RNA samples from COVID-19 patients (n = 70; conformed by RT-PCR) and healthy donors (n = 30) were used for clinical verification, and the accuracy rate for the RPA assay was 100%. The RPA results were 100% consistent with those of RT-PCR (Table 4), and with the advantage of a shorter assay time (3.39 min vs. 11.17 min) ( Figure 5). More importantly, boxplot and ROC analyses showed that the Cq value from COVID-19 patients was significantly different from that of healthy controls, and the developed RPA assay differentiated COVID-19 patients from healthy controls with a sensitivity of 100% at a specificity of 90% ( Figure 4). The most important thing is that the established detection method can effectively deal with the detection of virus variant strains, and can avoid missing detection (Table 2). It should be noted that, for RPA amplification in this study, fluorescence signals were collected every 30 s; therefore, 30 s was set as

| CONCLUSION
In summary, we developed an accurate, rapid method for COVID-19 and influenza diagnosis based on an RPA approach. The assay displayed a sensitivity of 100% for differentiating COVID-19 and influenza patients from healthy controls when the specificity was at a specificity of 96.67%, 96.67%, and 100% for N, ORF1ab and E gene, and 100%, 100%, and 90% for M, HA, and PA gene, respectively. The assay could potentially be used for screening COVID-19 and influenza or monitoring infections as an alternative method to RT-PCR or LAMP in low-resource settings.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.